The object of this research is to increase our understanding of the mechanism of RNA polymerase. We are using various spectroscopic techniques (circular dichroism, absorption spectra, fluorescence, electron paramagnetic resonance, and laser Raman spectroscopy) to study the conformation of RNA polymerase, both the core enzyme and holo enzyme, in order to assess the role of sigma factor. In addition to the intrinsic spectroscopic properties of the protein, various probes are bieng used - rifampicin, modified rifamycins with spin and fluorescence labels attached, and dyes such as Congo Red. These physical techniques and probes will be used to follow the reaction of polymerase with DNA to determine the kinetics of binding and the conformational state of the enzyme and DNA as a function of temperature, ionic strength, and nature of the template. Chemical modification procedures on the intact enzyme and isolated subunits will be used to probe the mechanism of DNA binding. Affinity- and photoaffinity-labelled analogues of nucleotides will be used to probe functional groups at the active site.